It is now well known that olives contain a number of bioactive compounds, particularly polyphenols; among these polyphenols, hydroxytyrosol is of outstanding biological significance in view of its antioxidant, antimicrobial and radical scavenging activity.
Hydroxytyrosol is present in olives and olive oil and has the following formula:

In co-pending patent applications EP07001791 and PCT/IB2008/000173, which are incorporated by reference, process and apparatus for the production of hydroxytyrosol from olives and/or olive oil extraction residues are described, which aim at the production of hydroxytyrosol-containing products or extracts to be used as a source of hydroxytyrosol in food, medical and cosmetic industries. The content of the above applications is hereto incorporated by reference.
Production of hydroxytyrosol from olives residues after oil extraction was and is actively investigated; an efficient extraction process could be very profitable especially because the major amount of hydroxytyrosol and hydroxytyrosol precursors initially present in the olives remains in the residues from olive oil production and only a minor part is found in the oil. Extra virgin olive oil normally contains 1-20 ppm of hydroxytyrosol.
The residues of olive oil extraction of interest for the present application exclude the olive tree leaves, because the leaves are removed before oil extraction. Moreover, hydroxytyrosol extraction from leaves faces different starting compositions (and therefore different extraction problems) than extraction from olives or olive residues.
The residues of olives as obtained from olive oil extraction processes of interest for the present application can be classified as:                pomaces, i.e. the solids containing residues of the pressing (in Spanish: orujo), of the three-phases process (orujo), or of the two-phases process, in which no water is added to the chopped olives in the centrifugation step (in Spanish: alperujo). Orujo, alperujo and defatted orujo contain a high amount of water (45% to 70%). Extraction residues also comprise orujillo, the olive dry solids, after orujo oil extraction that contains less than 15% water and is nearly free from oil residues.        
The green olives extracts of the invention are preferred to the extracts of residues of olive oil production in view of their greater amount of hydroxytyrosol and of the reduced content of hydroxymethylfurfural.
For the purposes of the present description the term “pomaces” or “solid residue” is designating both “orujo”, “defatted orujo” “orujillo” and “alperujo”. Preferred starting materials for the present invention are olives, more preferably green olives, and pomaces.
It is well known that several sensory properties are elicited by olive polyphenols in extra virgin and virgin olive oils. The sensory aspect of these olive oils has great repercussions on its acceptability by consumers. Some phenols mainly elicit the tasting perception of bitterness; however, other phenolic molecules can stimulate the free endings of the trigeminal nerve located in the palate and also in the gustative buds giving rise to the chemesthetic perceptions of pungency, astringency and metallic attributes.
Oleuropein, the phenolic compound that makes the fruit of the olive bitter, is water-soluble rather than fat-soluble, and it gets poorly transferred into the oil when the fruit is pressed, thus average content of oleuropein in extra virgin and virgin olive oils is from 1 ppb to 11 ppm.
Oleuropein is present in olives and olive oil and has the following formula:

Nevertheless, a number of oleuropein related compounds are more oil soluble than oleuropein itself, and do end up at a higher content in the oil, in the form of isomer (or isomers) of oleuropein aglycone (e.g.: aldehydicic form of oleuropein aglycone, AOA), and dialdehydic form of elenolic acid linked to hydroxytyrosol or tyrosol, respectively named 3,4-DHPEA-EDA and p-HPEA-EDA, that are the olive polyphenols mainly responsible for the bitter taste according to Gutierrez-Rosales and co-authors, J. Agric. Food Chem. 2003, 51, 6021-6025.
Oleuropein aglycone (e.g.: aldehydic form of oleuropein aglycone) is present in olive oil and in crushed olives during oil extraction process and has the following formula:

It is known to carry out acid hydrolysis of the pomaces (or of vegetation water) to have the cleavage of the ester bond in the oleuropein molecule and obtain hydroxytyrosol.
U.S. Pat. No. 6,361,803, which is incorporated by reference, discloses extraction of hydroxytyrosol (and other compounds) by neutral or acid hydrolysis of olive pulp residues at reflux for one hour (ex.12). The extracted water solution is loaded on an absorption XAD-7 column that is eluted with methanol to recover the extracted hydroxytyrosol. U.S. Pat. No. 6,361,803 requires the use of organic polar solvents, to recover hydroxytyrosol with a minimum purity grade, in addition (ex.12) freeze precipitation of some impurities from the methanol solution is necessary. Polar aqueous solvents are selected among methanol, ethanol, acetonitrile or acetone, while polar organic solvents are selected, for example, among esters, amides, dimethyl sulfoxide, dioxane, DMF and their mixtures. Most of these solvents are toxic and very difficult to completely eliminate from the desired hydroxytyrosol product. Accordingly, traces of the utilized solvents will be found in the final product even after several purification steps, thus rendering the hydroxytyrosol obtained according to this process not suitable for a safe application in the alimentary, cosmetic and pharmaceutical field. Moreover, the final product is not suitable for use in fortifying foods, particularly edible oils, without use of ethanol and acetic acid as additives to the extract to obtain a stable food product: the resulting food product (oil plus hydroxytyrosol containing extract, ethanol and acetic acid) is not acceptable in the food industry.
WO 2004/005228, which is incorporated by reference, discloses hydrolysis at room temperature of vegetation water obtained from olive oil extraction by incubation of the acidified vegetation water for at least two months and preferably 6-12 months until at least 50% (preferably 90%) of the oleuropein originally present in the vegetation water has been converted to hydroxytyrosol. The incubated vegetation water is extracted with an organic solvent, for example ethyl acetate, or it is contacted with a supercritical fluid (CO2), to produce a fraction rich in hydroxytyrosol. The main problems of this process are the very long time required for the incubation of the acidified vegetation water and the use of organic solvents, that should be avoided, particularly when the final product obtained is to be used in the alimentary, cosmetic and pharmaceutical field.
U.S. Patent Application Publication 2004/0102657, which is incorporated by reference, discloses acidic hydrolysis with a steam explosion process at high temperatures (about 190-220° C.). The obtained solution is firstly partially purified on a column with non activated ion exchange resin and subsequently loaded on a XAD non-ionic column, from where the hydroxytyrosol is eluted with methanol or ethanol. The process results in a poor yield in hydroxytyrosol, considering the process in its entirety.
EP 1623960, which is incorporated by reference, 0, discloses a process of recovery of hydroxytyrosol, and tyrosol, from alpechin by means of filtration in a complicated plant consisting of three units (Ex. 1) and subsequent separation. The tyrosol is then oxidated to hydroxytyrosol in a protic solvent (alcohol or water) to obtain a final product that is semi-synthetic. Additionally, EP 1623960 discloses that concentration by nanofiltration and reverse osmosis of vegetation waters (alpechin) are carried out at neutral or alkaline pH (claim 4). This process has two important problems. A first drawback is that hydroxytyrosol degradation increases at neutral and alkaline conditions giving unwanted products that are difficult to remove, and, in addition, due to the fact that no acid hydrolysis is carried out the oleuropein content should be still high. Secondly, the obtainable concentration factor is poor, thus the maximum concentration allowed for hydroxytyrosol according Ex-1 (see table 1) is in the range from 1.2 to 1.6 g/L.
U.S. Patent Application Publication 2004/0176647, which is incorporated by reference, discloses an extraction process of phenols from alperujo under stirring at 180-240° C. in autoclave, in water. No acid is added, but the thermal treatment results in a “liberation of acetyl groups” and in an alleged consequent reduction of the pH (page 3, first paragraph). Nevertheless, at the pHs produced in the described conditions hydrolysis of oleuropein is far to be complete, meaning two things, lower hydroxytyrosol yields and remaining hydrolysed oleuropein, that as we discuss above is causing non-pleasant taste in food products containing such extracts, that are thus not suitable for food applications. The products are tyrosol and hydroxytyrosol, that are separated by HPLC with sulphuric acid/acetonitrile eluent.
Summarizing, the above mentioned techniques are either too long or too complex, or too harsh, or all of the above; this results in that the amount of remaining oleuropein, i.e. the amount that is not hydrolyzed, and/or the amount of the hydrolysis by-products such as hydroxymethylfurfural is high enough to make difficult the subsequent purification steps.
Obtaining a good source of hydroxytyrosol is very important for the fortification of foods, especially of edible oils. Extracts should not transfer to oils any bitter polyphenolic substances, however, the health beneficial compounds, namely hydroxytyrosol, are always associated to bitter polyphenols in the known extracts.
Frank and co-authors in J. Agric. Food Chem. 2001, 49, 231-238 disclose a procedure called “taste dilution analysis” in order to point out the sensory threshold of bitter for oleuropein derivatives. Bitterness was assessed by preparing serial dilutions of samples in water and then tasting them according to increasing concentration, until the concentration at which the diluted sample can be differentiated from water as judged in a triangle test is found.
It was also shown that at least for these compounds there is no direct correlation of bitterness with the absorbance at 225 (the K225 value): Mateo et al. J. Am. Oil Chem. Soc. 2004, 81, 71-75, verified the correlation between the aldehydicic form of oleuropein aglycone (obtained by hydrolysis of oleuropein with β-glucosidase from almonds purchased from Sigma) and bitterness.
Andrewes et al., J. Agric. Food Chem. 2003, 51, 1415-1420, assessed the relationship between polyphenols and olive oil pungency; p-HPEA-EDA was the key source of the burning sensation found in many olive oils.
In 2005, Beauchamp and co-authors, Nature 2005, 437, 45-46, measured the pungent intensity of p-HPEA-EDA isolated from different extra virgin and virgin olive oils confirming this molecule is the principal agent in extra virgin and virgin olive oils responsible for throat irritation.
Additionally, the bitter and pungent polyphenols present in olive oil, are also presents in olives and are also responsible for their bitterness and pungency, those being the ones that best correlate with the bitterness attribute the oleuropein and the aldehydic form of oleuropein aglycone. The crushing of olives to prepare olive oil or olive extracts produces the hydrolysis of oleuropein by endogenous β-glucosidase rendering oleuropein aglycone as transient specie that forms the aldehydic form of oleuropein aglycone. Thus, the use of olive extracts containing oleuropein and/or the aldehydic form of oleuropein aglycone can modify the taste of foods in which the aforementioned extract is incorporated, resulting in a lower acceptance by consumers.
Summarizing, any way set up in order to increase antioxidant capacity using techniques based on fortification of foods with olive polyphenols, should not alter food natural organoleptic characteristics, nor increase the amount of the bitter tasting olive polyphenols, otherwise incurring with the subsequent alteration of organoleptic properties of the food, thus causing non-pleasant taste due to excessive bitterness, pungency and/or astringency, and subsequently causing rejection of the fortified food from many consumers.
It is known to add polyphenols and hydroxytyrosol to food products.
U.S. Pat. No. 6,942,890 which is incorporated by reference, discloses a method of fortifying food products adding to such products solid matter derived from olive fruits, resulting in an increase of the level of antioxidants, particularly of olive polyphenols.
It has also to be noted that all the previously mentioned techniques allow to obtain a fortified food with an increased content of total olive polyphenols but with a low content in hydroxytyrosol with respect to the total olive polyphenols content, due to the fact that most of the polyphenols incorporated to the fortified food are secoiridoids, oleuropein related compounds as oleuropein aglycone (AOA), 3,4-DHPEA-EDA and p-HPEA-EDA, at a higher content than hydroxytyrosol, and that those olive polyphenols are mainly responsible for the bitter taste and pungency of the fortified oil. The incorporation of these secoiridoids, able to produce undesirable changes in the organoleptic properties of the fortified food, is in fact directly related with the source of olive polyphenols used in all the previously mentioned techniques, which in fact present a low content in hydroxytyrosol and/or a low purity degree.
A few studies addressed the bioavailability of polyphenolic compounds in olive showing that the absorption of olive oil phenolics is probably larger than 55-66 mol %, and that the absorption of hydroxytyrosol is dose-dependent, suggesting that olive oil phenolics are absorbed from the intestine, that tyrosol and hydroxytyrosol are incorporated in lipoprotein fractions, and that hydroxytyrosol is excreted in urine as glucuronide conjugate. The incorporation of phenolic compounds from olive oil in LDL particles has been proposed as the mechanism by which olive phenolics may protect LDL particles from peroxidation.
The EFSA Panel on Dietetic Products, Nutrition and Allergies (NDA) concludes that a cause and effect relationship has been established between the consumption of olive oil polyphenols (standardized by their content of hydroxytyrosol and its derivatives) and protection of blood lipids from oxidative stress.
According to Regulation (EC) No 1924/2006 of the European Parliament and of the Council of 20 Dec. 2006 on nutrition and health claims made on foods, a claim that a food is sugars-free, and any claim likely to have the same meaning for the consumer, may only be made where the product contains no more than 0.5 g of sugars per 100 g or 100 ml.